The invention relates to compositions, systems, and methods for detecting molecular species using nucleic acids.
Various types of systems have been used to detect the presence of a particular chemical or molecule in a complex sample. For example, antibodies are used to detect the presence of a protein in a sample, and DNA microarray chips have been used to identify genes and study gene expression. Most existing molecular detection systems are designed to detect the presence of a single type or single category of target molecule. In the case of antibody detection, existing systems are typically limited to detecting only a subset of a type of molecule.
Recently, it has been shown that RNA and DNA aptamers can substitute for monoclonal antibodies in various applications (Jayasena, xe2x80x9cAptamers: an emerging class of molecules that rival antibodies in diagnostics.xe2x80x9d Clin. Chem., 45(9):1628-50, 1999; Morris et al., xe2x80x9cHigh affinity ligands from in vitro selection: complex targets.xe2x80x9d Proc. Natl. Acad. Sci., USA, 95(6):2902-7, 1998). The relatively fast selection process of the specific aptamers and the inexpensive synthesis makes the aptamer useful alternatives for monoclonal antibodies. These nucleic acids can be easily synthesized, readily manipulated, and can be stored for over long time. These benefits make nucleic acids more attractive biotechnology tools than their counterpart of proteins, antibodies. Additionally these nucleic acid probes can also be labeled by radioisotope, biotin, or fluorescent tags and can be used to detect targets under various conditions. An increasing number of DNA and RNA aptamers that recognize their non-nucleic acid targets has been developed by SELEX and has been characterized (Gold et al., xe2x80x9cDiversity of Oligonucleotide Functions,xe2x80x9d Annu. Rev. Biochem., 64:763-97.1995; Bacher and Ellington, xe2x80x9cNucleic Acid Selection as a Tool for Drug Discovery,xe2x80x9d Drug Discovery Today, 3(6):265-273,1998).
The invention relates to new compositions, systems, and methods for simultaneously detecting the presence and quantity of one or more different compounds in a sample using novel nucleic acid sensor molecules. Nucleic acids have previously been shown to be capable of specifically binding with high affinity to non-nucleotide target molecules, such as proteins, small organic molecules, or inorganic molecules. These nucleic acids are commonly referred to as aptamers. An aptamer can be either an RNA or a DNA composed of naturally occurring or modified nucleotides.
In the new compositions, standard aptamers are bioengineered such that binding of a bioengineered aptamer to a target molecule causes a change in the conformation of the bioengineered aptamer. Furthermore, one or more reporter moieties or groups are included in the bioengineered aptamers such that the change in bioengineered aptamer conformation results in a detectable change of a physical property of the reporter group (or the engineered aptamer itself). These bioengineered aptamers are referred to herein as aptamer beacons.
Aptamer beacons having binding regions configured to bind to different target molecules can be used in various detection methods and systems. For example the new aptamer beacons can be used in solution-based assays, or can be attached to a solid support, e.g., at different predetermined points in a one or two-dimensional array, for use in solid-based assays. The aptamer beacons or aptamer beacon arrays are then exposed to the sample, such that target molecules in the sample bind to their respective aptamer beacons. The presence of bound target molecules can be detected by measuring a change in a physical property of the aptamer beacon reporter group, e.g., by observing a change in fluorescence efficiency of the aptamer beacon.
To assist in analyzing the sample, the new detection systems can include pattern recognition software. The software compares the target molecule binding pattern corresponding to the unknown sample with binding patterns corresponding to known compounds. From these comparisons, the software can determine the composition of the sample, or deduce information about the source of the sample.
The systems can be used to detect the existence of characteristic compounds, or xe2x80x9cmolecular fingerprints,xe2x80x9d associated with certain chemicals or conditions. For example, the systems can be used for human drug testing by detecting the presence of metabolites of particular drugs. The systems can also be used to infer the existence of a disease (e.g., cancer) by detecting the presence of compounds associated with the disease state, or for pollution monitoring by detecting compounds characteristic of the discharge of certain pollutants. Numerous other applications are also possible.
In general, the invention features an aptamer beacon that binds to a non-nucleic acid target molecule and that includes an oligonucleotide including a loop portion, a first segment, and a second segment complementary to the first segment, wherein the first and second segments form a stem portion when hybridized together; a binding region formed by the oligonucleotide and configured to bind to the non-nucleic acid target molecule; a first reporter moiety, e.g., a fluorophore, attached to the first segment; and a second reporter moiety, e.g., a chemical quencher, attached to the second segment, wherein the first and second reporter moieties interact to produce a detectable signal when the distance between them is changed; wherein binding of the target molecule to the binding region breaks base pair bonds in the stem portion, causing a change in conformation of the aptamer beacon that separates the first and second segments, thereby altering the distance between the first and second reporter moieties, and producing a detectable signal.
A conformational change in an aptamer beacon is an alteration in the secondary and/or tertiary structure of the oligonucleotide that makes up the aptamer beacon. A conformational change typically results in the addition and/or deletion of basepairing interactions in the between alternate forms of the aptamer beacon.
In these new aptamer beacons, when the first reporter moiety is a fluorophore and the second reporter moiety is a chemical quencher, the quencher quenches the fluorophore when the first and second segments are hybridized together to form the stem portion, and wherein binding of a target molecule to the binding region breaks base pair bonding in the stem portion, causing the first and second segments to separate and the fluorophore to separate from the chemical group, thereby ending the quenching and enabling the fluorophore to emit detectable fluorescence.
In these aptamer beacons, the binding region can be located entirely or partially within the loop portion, the stem portion, or at least partially in both. In addition, the first and second reporter moieties can be an enzyme and a corresponding ligand, and the first and second segments can include 4, 5, 6, or 7 nucleotides each.
The invention also features an aptamer beacon that binds to a non-nucleic acid target molecule and that includes an oligonucleotide including a first segment, a second segment, and a third segment located between the first and second segments, wherein the first and second segments form a complex, e.g., a hybrid duplex or other secondary or tertiary structure, when the aptamer beacon is not bound to the target molecule; a binding region formed by the aptamer beacon when contacting the target molecule; a first reporter moiety attached to the first segment; and a second reporter moiety attached to the second segment, wherein the first and second reporter moieties interact to produce a detectable signal when the distance between them is changed; wherein binding of the target molecule to the binding region breaks base pair bonds in the complex causing a change in conformation of the aptamer beacon that alters the distance between the first and second reporter moieties, and producing a detectable signal.
In these aptamer beacons, the first reporter moiety can be an energy absorbing moiety and the second reporter moiety can be a fluorescence emitting moiety, such that when the first and second reporter moieties are in sufficiently close proximity, the absorbing moiety allows an energy transfer between the moieties, thereby allowing the emitting moiety to emit fluoresce; and wherein binding of a target molecule to the binding region causes the first and second segments to hybridize together.
In another aspect, the invention features a device for simultaneously detecting the presence of a plurality of different, non-nucleic acid target molecules in a sample. The devices include: a solid support; and a plurality of different aptamer beacons bound to the support, each aptamer beacon having a first end attached to the support, and a binding region that binds to a specific non-nucleic acid target molecule, wherein the binding regions of different aptamer beacons bind to different target molecules. In these devices, the solid support can be a glass surface to which the first ends of the aptamer beacons are covalently bound. In addition, the solid support can be a planar surface, and the aptamer beacons can be distributed on the planar surface in a two-dimensional array. Spots of identical aptamer beacons can be located at different points in the two-dimensional array.
The binding region of at least one of the aptamer beacons in the device can be configured to bind to a non-nucleic acid target molecule selected from the group consisting of a protein, a steroid, and an inorganic molecule. The aptamer beacons can comprise RNA, DNA, modified RNA, modified RNA, or a combination thereof. In addition, each aptamer beacon can comprise a reporter group, such as a fluorophore, for signaling binding of a target molecule to the binding region.
The invention also features a method of detecting the presence or absence of one or more different target molecules in a sample, by obtaining a plurality of the new aptamer beacons; contacting the sample to the aptamer beacons, such that any target molecules in the sample can bind to corresponding binding regions of the aptamer beacons; and detecting the presence of target molecules bound to the aptamer beacons. The aptamer beacons can be in a liquid, or can be bound to a solid support, such as a particle or a plate. In some embodiments, the aptamer beacons emit fluorescent radiation when excited by evanescent waves.
In this method, different spots, each spot including a plurality of identical aptamer beacons, can be distributed on the solid support in a predetermined array, and the method can further include comparing a fluorescence pattern of the sample to known fluorescence patterns, e.g., with a computer program, disposed on a computer readable medium, that includes instructions for causing a processor to compare the fluorescence pattern of the sample to a library of known fluorescence patterns; and select the combination of known fluorescence patterns that most closely matches the fluorescence pattern of the sample.
The detecting step can also include detecting a change in the Raman emission frequencies of an aptamer beacon caused when a target molecule binds to the aptamer beacon.
In another aspect, the invention features a computer program, disposed on a computer-readable medium, for analyzing the output of an assay that determines the composition of a sample and deduces the presence or absence of known abnormal conditions, the computer program including instructions for causing a processor to: compare the assay output, e.g., an image, to a library of known outputs corresponding to subjecting samples of known composition to the assay; select a combination of known outputs that most closely matches the assay outputs; compare any deviation between the sample output and the combination of known outputs to a library of known deviations, the known deviations being caused by known abnormal conditions; and deduce the presence or absence of known abnormal conditions. For example, the known abnormal conditions can include the presence of abnormal compounds in the sample, and the presence of normal compounds in abnormal quantities.
The invention also features a method for analyzing the output of an assay that determines the composition of a sample and deduces the presence or absence of known abnormal conditions by comparing the assay output to a library of known outputs corresponding to subjecting samples of known composition to the assay; selecting a combination of known outputs that most closely matches the assay outputs; comparing any deviation between the sample output and the combination of known outputs to a library of known deviations, the known deviations being caused by known abnormal conditions; and deducing the presence or absence of known abnormal conditions.
In yet another aspect, the invention features a device for detecting the presence of a target molecule in a sample. The device includes a solid support; and a plurality of different aptamer beacons bound to the support, each aptamer beacon having a first end attached to the support, and a binding region that binds to a specific enantiomer of the target molecule, wherein the binding regions of different aptamer beacons bind to different enantiomers of the target molecule.
Furthermore, the invention includes a device for detecting the presence of a target in a sample. The device includes a solid support; and a plurality of different aptamer beacons bound to the support, each aptamer beacon having a first end attached to the support, and a binding region that binds to a specific binding site of the target, wherein the binding regions of different aptamer beacons bind to different binding sites. For example, the target can be an antigen, and the different binding sites can be different epitopes of the antigen, or the target can be a bacteria, and the different binding sites can be different surface proteins of the bacteria.
The invention further features a system for simultaneously detecting the presence of a plurality of different non-nucleic acid target molecules in a sample. The system includes a solid support (optional); a plurality of different aptamer beacons, optionally bound to the support, each aptamer beacon having a first end attached to the support, a binding region that binds to a specific non-nucleic acid target molecule, the binding regions of different aptamer beacons binding to different target molecules; and a detection system that detects the presence of target molecules bound to aptamer beacons, the detection system including a radiation source, e.g., a laser, and a detector. The system can further include an analyzer for determining the presence of target molecules in the sample based on the output of the detection system. The analyzer can also include a computer processor programmed to compare the output of the detection system to a library of known outputs corresponding to exposing samples of known composition to the aptamer beacons on the solid support; and select a combination of known outputs that most closely matches the assay outputs. The computer processor can be further programmed to compare any deviation between the output of the detection system and the combination of known outputs to a library of known deviations, the known deviations being caused by known abnormal conditions; and deduce the presence or absence of known abnormal conditions.
In yet another aspect, the invention features a method or system for simultaneously detecting the presence or absence of one or more different target molecules in a sample using a plurality of different species of aptamer beacons, wherein each species of aptamer beacons has a different reporter group, a binding region that binds to a specific non-nucleic acid target molecule, and wherein the binding regions of different aptamers bind to different target molecules; and a detection system that detects the presence of target molecules bound to aptamer beacons, the detection system being able to detect the different reporter groups. The method can also be carried out with a plurality of identical aptamer beacons. For example, each aptamer can include a reporter such as a molecular beacon that changes fluorescence properties upon target binding. Each species of aptamer beacon can be labeled with a different fluorescent dye to allow simultaneous detection of multiple target molecules, e.g., one species might be labeled with fluoroscein and another with rhodamine. The fluorescence excitation wavelength (or spectrum) can be varied and/or the emission spectrum can be observed to simultaneously detect the presence of multiple targets.
The fluorescence measurement can be performed with a number of different instruments, including standard fluorescence spectrophotometers, or in a small volume using a high-intensity source, such a laser, high-efficiency light collection optics, such as a high-numeric aperture microscope objective, and a high-efficiency low-noise detector, such as photo-multiplier tube, a photodiode or a CCD camera.
The method can further include a computer program that includes instructions for causing the processor to compare the measured fluorescence emission or excitation spectrum with the known spectrum of each of the individual dyes to quantitatively determine the concentration of each of the target molecules in the solution.
Different aspects of the invention may include one or more of the following advantages. The aptamer beacon-based detection systems allow the detection of a plurality of different compounds simultaneously, or high sensitivity detection of a single target in a plurality of different ways. Unlike antibodies, which are selected in an organism, the aptamers can be selected in vitro, e.g., in a test tube. This allows detection of target molecules that are toxic or immunologically inert. Unlike standard aptamers, the new aptamer beacons transduce the aptamer:target binding interaction into a detectable change in physical properties of the aptamer beacon.
Furthermore, the aptamer beacons in the detection systems have high affinities for their target molecules, allowing ultra-sensitive detection. As a result, the systems are highly specific, and can distinguish molecules that differ by as little as a single methyl or hydroxyl group.
The systems also allow rapid analysis of a sample (as quickly as a few minutes), facilitating detection of unstable compounds. In addition, the reagents used in the assay are inexpensive, and the chemistry involved in performing the assay is easily automated.
The detection systems can be used in a variety of applications, including drug testing, high-sensitivity testing for the presence of bacteria or antigens, pollution monitoring, and testing for the presence or absence of a disease.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.